The Molecular Pharmacological Characteristics Of A New K_ATP Channel Opener, Iptakalim, Highly Specific For Opening SUR2B/Kir6.1 Channel

ATP-sensitive potassium(KATP) channels, widely distributed in many tissues, play a variety of physiological and pathological roles by coupling the metabolic state to cellular electrical activity. KATP channels usually remain closed under normal physiological conditions but they can be activated when cell metabolism is inhibited under hypoxia or ischemic stress, in which condition the concentration of intracellular energy substances change markedly(ATP concentration decreased and ADP concentration increased), resulting in efflux of potassium ions and membrane hyperpolarization. The decreased cell excitability then will participate in a variety regulating functions. Therefore, the KATP channels play an important role in the occurrence and development process of many cardiovascular diseases such as hypertension. So, as a new drug target, the KATP channel has caused extensive concern.Molecular biology studies have shown that KATP is a hetero-octameric complex(Kir6.x/SUR) 4 consisting of inwardly rectifying potassium channel subunit(Kir) and sulfonylurea receptor(SUR) subunit at the ratio of 1:1. The Kir subunits participating in KATP composition are mainly Kir6.x(including Kir6.1, Kir6.2) and Kir1.1. Kir6.x widely exists in various organs and tissues, while Kir1.1 mainly expresses in the kidney. Kir6.x subunit mainly determines the electrophysiological properties of KATP channels. SUR, as the regulatory subunit, surrounding in the external of Kir subunits, including three subtypes: SUR1, SUR2 A and SUR2 B. They are considered to be the targets of channel modulators(suchas nucleotide, KCOs and sulfonylureas inhibitors etc.). Different subtypes of subunits share gene and amino acid identity, but they also have obvious differences, which underlying the molecular mechanism of the selective effects of drugs. Native KATP channels in various tissues are believed to be composed of differential subtypes of Kir6.x and SUR subunits. Coexpression of SUR1/Kir6.2, SUR2A/Kir6.2, SUR2B/Kir6.2 has been reported to reconstitute pancreatic β-cell and some neurons, cardiac and skeletal muscle, non-vascular smooth muscle KATP channels, respectively. A variety of studies has indicated that the vascular smooth muscle KATP channel is constituted at the molecular level by SUR2B/Kir6.1. In addition, there are reports indicating that the close resemblance between mito KATP channels and SUR1/Kir6.1 combination in their pharmacological profiles. As the molecular diversity of different subtypes of KATP channels, they have their own unique physiology, pathophysiology and pharmacology characteristic. Therefore, it is possible to develop the tissue-selectivity KATP channel opener with light side effects.Iptakalim is a new KATP channel opener with new chemical structure differing from other KCOs, and it is designed and synthesized by ourself. The drug has beening studied in phase III multicenter clinical trial and our previous studies have established that iptakalim has unique advantages in pharmacology and treatment. It can selectively dilate resistance vessels vascular and has few side effects in the treatment of hypertension; moreover, it is only effectual in hypertensive patients and without effects on people with normal blood pressure. By using glibenclamide and gene knockout models, we have confirmed the major effects of iptakalim such as vasodilation, the protection and improvement of organ damage and congestive cardiac remodeling are all closely related with the activation of KATP channels in vascular smooth muscle and endothelial cells.Previous studies have used fluorescence-based assays to examine the capacity of iptakalim and natakalim to activate different types of recombinant KATP channels and some patch clamp experiments have been done to confirm the subtype-selective of iptakalim on the KATP channels. However, these studies were limited to the low sensitivity of fluorescence responses using Di BAC4(3) and cells used in the patch-clamp experiments some natural cells or HEK293 cells only expressing three subtypes of KATP channels. Therefore, the subtype-selective characteristics of iptakalim and natakalim on KATP channels are far from fully understood. In addition, Ipt has showed selectivity of the hypertension state, suggesting its action may related with the energy metabolism of cells, but this also lack of direct experimental evidences. In this study, using Xenopus oocyte as the heterologous expression vector, we expressed all six subtypes of KATP channels and and designed experiments by using two-electrode voltage clamp technique(TEVC), which a method combined molecular biology, electrophysiology and pharmacology. We systematiclly studied the subtype selectivity of iptakalim and natakalim on KATP channels and their regulatory role in energy metabolism, as well as the molecular pharmacological mechanisms of their actions, such as the definition of their binding sites on KATP channels and several important amino acid sites in the regulation of intracellular energy material. This study will give direct and comprehensive understanding of the pharmacological characteristics and molecular mechanisms of the subtype-selective effects of the two new KATP channel openers iptakalim and natakalim. The First Part: Functional characteristics of different subtypes of KATP channels expressed in Xenopus oocytes.Using Xenopus oocyte as the expression vector, we expressed all six subtypes of KATP channels and then used two electrode voltage clamp technique, to study of the six subtypes of KATP channels in pharmacology and physiology(mainly is the sensitivity to metabolic inhibition) functional characteristics systematiclly.The control oocytes(water-injected) had no responses to Na-azide or these openers and blockers, indicting there is no expression of KATP in oocyte, So it be used as a heterologous expression vector; and, Kir6.x or SUR subunit alone cannot be expressed in the oocyte membrane to form functional channels.Pharmacological characteristics of different subtypes of KATP channels.The results indicate that SUR1/Kir6.x could be stimulated by diazoxide but not by pinacidil; SUR2A/Kir6.x were stimulated by pinacidil but not by diazoxide; whereas SUR2B/Kir6.x can be stimulated by both of them. Six subtypes of KATP channels all can be inhibited by glibenclamide and Ba2+ significantly; 5-HD highly selectively block the SUR1/Kir6.x KATP channels with a stronger inhibition effect on SUR1/Kir6.1; PNU-37883 selectively blocked SUR/Kir6.1 and SUR2B/Kir6.2 subtype KATP channels.Different subtypes of KATP channels are indeed significantly different in their metabolic sensitivities. Na-azide can inhibit the cellular energy metabolism in oocytes, and the intracellular ATP content decreased markedly, while the ratio of ADP/ATP increased significantly. Different subtypes of KATP channels are indeed significantly different in their metabolic sensitivities, which are mainly determined by the subtype of SUR subunits and affected by the Kir6.x subunit. The Second Part: Subtype-selectivity of the new KATP channel opener iptakalimWe tested the effects of iptakalim and natakalim on the whole-cell currents of different subtypes of KATP channels and provided direct experimental evidence for their selectivities on all subtypes of KATP channels. Furthermore, we also observed the selective modulation effects of iptakalim and natakalim on different subtypes of KATP channels when cell metabolism is inhibited which was induced by 3m M Na-azide.The selectivity effects of iptakalim and natakalim on different subtypes of KATP channels. The six subtypes of KATP channels remain closed under normal physiological conditions and Ipt and Nat can selectively activate the SUR2B/Kir6.1 channel, but had no significant effects on other subtypes. When the cellular energy metabolic was inhibited by Na-azide, the SUR2B/Kir6.1 subtype KATP channel is pre-activated. And then, the activation effects of Ipt and Nat on SUR2B/Kir6.1 chan-nels were more significant compared with the effects of resting state.Inhibition of energy metabolism(or combined with KCO) could pre-activate the SUR2A/Kir6.1 and SUR1/Kir6.1 subtypes KATP channels, whereas Ipt and Nat had no significant effects on them; Howerer, they did has certain inhibitory effects on the three SUR/Kir6.2 combinations when they were pre-activate by cellular metabolic inhibition. The Third Part: Molecular mechanisms of the subtype-selectivity of iptakalimAs the new type of KCOs, which subunit is the binding site of iptakalim and natakalim, SUR or Kir6.x? Whether they have direct effects on Kir subunits? Previous studies have showed that the activation effects of iptakalim and natakalim on SUR2B/Kir6.1 channel were depend on the cellular metabolic state, and then whether the binding and/or hydrolysis of intracellular nucleotides(Mg ATP and Mg ADP) on SUR2 B subunit would affect the activation effects of these drugs? Given these issues, we first studied the effects of iptakalim and natakalim on the independently expressed KATP subunits such as Kir6.1ΔC45, Kir6.2ΔC36 and Kir1.1, and then using the SUR2 B mutant models, we further revealed two important nucleotide binding and/or hydrolysis sites which associated with the the regulation of drug effects by intracellular energy material.When the retention signal sequence was removed from the C-terminal of Kir6.x, individual Kir6.x subunits(Kir6.1ΔC45 and Kir6.2ΔC36) can form functional channels on the membrane of Xenopus oocyte. Iptakalim and natakalim had no effects on Kir6.1 and Kir1.1 subunit, indicating that Ipt and Nat activated the SUR2B/Kir6.1 KATP channels by binding to SUR2 B subunit.No matter whether the cell energy energy metabolic was inhibited or not, Ipt and Nat had certain inhibition effects on the Kir6.2ΔC36 subunit channel, and these inhibitory effects of the drugs were similar with the effects of them on the three subtypes of SUR/Kir6.2 channels which were pre-activated by metabolic inhibition. S o, these results suggested that Ipt and Nat inhibited the SUR/Kir6.2 subtype KATP channels by binding to Kir6.2 subunit.The NBDs of SUR2 B subunit have two important nucleotide binding and/ or hydrolysis sites as K707 and K1348. When they were single-point mutated, Ipt and Nat loss their activation effects on SUR2B/Kir6.1 channels under normal physiological conditions, but when the cellular energy metabolic was inhibited, they did has certain activation effects on these channels, whereas these effects were greatly reduced compared with the wild-type channel. This suggested that these two important nucleotide(Mg ATP and Mg ADP) binding and/or hydrolysis sites were associated with the the activation effects of Ipt and Nat on the SUR2B/Kir6.1 subtype KATP channels, which may due to the allosteric effects of drugs and intracellular energy material in regulating KATP channel activity and thus proved a link between cellular metabolic states to drug effects. The main conclusions of this study: 1. Under normal physiological conditions, Ipt and Nat can selectively activatethe SUR2B/Kir6.1 subtype channels, and when the cellular energy metabolic wasinhibited, the activation effects of Ipt and Nat on SUR2B/Kir6.1 channels wereenhanced significantly. 2. Ipt and Nat had no effect on Kir6.1 and Kir1.1 subunit, indicating that they activate theSUR2B/Kir6.1 subtype KATP channels by binding to SUR2 B subunit. 3. The two important nucleotide binding and/or hydrolysis sites K707 and K1348 in NBDsof SUR2 B subunit were associated with the the activation effects of Ipt and Nat on theSUR2B/Kir6.1 subtype KATP channels, which proved a link between cellular metabolicstates and drug effects. 4. When the channels were pre-activated by metabolic inhibition, Ipt and Nat had certaininhibition effects on three SUR/Kir6.2 combinations, and these effects were similarwith that of drugs on Kir6.2 subunit(Kir6.2ΔC36), suggesting that Ipt and Natinhibited the SUR/Kir6.2 subtype channels by binding to Kir6.2 subunit.In summary, using the heterologous expression system and electrophysiological technique, we systematically studied the selective effects of iptakalim and natakalim on six subtypes of KATP channels and elucidated the possible molecular mechanisms. This study provides direct experimental evidences and comprehensive understanding of the subtype-selective pharmacological characteristics of the new KCOs,as well as their roles when the channels are pre-activated by metabolic inhibition. In addition, we provide some clues for exploring the molecular basis of the relationship between the intracellular energy substances and the drug effects.